Radiation/chemotherapy sensitizer to be used for intratumoral local injection and for controlled release of hydrogen peroxide with hydrogel as carrier

ABSTRACT

The present invention provides a radiation sensitizer or anti-cancer chemotherapy sensitizer that prevents the decomposition of hydrogen peroxide for a longer period of time, and that maintains its effect. The radiation sensitizer or anti-cancer chemotherapy sensitizer according to the present invention is characterized by using hydrogen peroxide together with a hydrogel containing a crosslinked gelatin gel.

TECHNICAL FIELD

The present invention relates to a sensitizer for enhancing atherapeutic effect of radiation or an anti-cancer agent on a tumor, andparticularly to a sustained-release radiation sensitizer or anti-cancerchemotherapy sensitizer injected into a tumor area upon irradiation withradiation or administration of an anti-cancer agent. The presentinvention further relates to an anti-cancer therapy using thesensitizer.

BACKGROUND ART

As a method for locally treating malignant tumors, radiotherapy issecond after surgical operations. Because radiotherapy can be applied toelderly patients and also enables normal organs and tissues to beconserved, the number of patients treated with this method has recentlybeen increasing very rapidly. However, linear-accelerator-generatedhigh-energy X-rays and electron beams that are currently used commonlyfor radiotherapy are low linear energy transfer (LET) radiation, whichhas a relatively low biological effect. Accordingly,linear-accelerator-based radiotherapy has little effect on tumors suchas malignant melanoma, various types of sarcoma, and glioblastomamultiforme. Because locally advanced cancers that have grown to severalcentimeters or more have many hypoxic cancer cells or contain largeamounts of antioxidative enzymes, such cancers are not only resistant toradiation but also often resistant to anti-cancer chemotherapy.

The present inventors previously confirmed that using hydrogen peroxidetogether with radiotherapy or anti-cancer chemotherapy is effective forsuch turners that are resistant to radiotherapy or anti-cancerchemotherapy (see, for example, Non-patent Literature (NPL) 1 to NPL 3.)

Further, the present inventors previously confirmed that using hydrogenperoxide together with hyaluronic acid or a salt thereof is mosteffective for intratumorally administering hydrogen peroxide, which ishighly irritant to the affected area and is prone to decompose in tumors(see, for example, Patent Literature (PLT) 1, and NPL 4 to NPL 7).

CITATION LIST PTL

PTL 1: WO2008/041514

PTL 2: JP2004-203829A

PTL 3: JP2005-325075A

NPL

NPL 1: Yasuhiro Ogawa at al., Mechanism of apoptotic resistance of humanosteosarcoma cell line, HS-Os-1, against irradiation, InternationalJournal of Molecular Medicine 12:453-458, 2003

NPL 2: Yasuhiro Ogawa et al., Apoptotic-resistance of human osteosarcomacell line HS-Os-1 to irradiation is converted toapoptotic-susceptibility by hydrogen peroxide: A potent role of hydrogenperoxide as a new radiosensitizer, International Journal of MolecularMedicine 12: 845-850, 2003

NPL 3: Yasuhiro Ogawa et al., Immunocytochemical characteristics ofhuman osteosarcoma cell line HS-Os-1: Possible implication in apoptoticresistance against irradiation, International Journal of MolecularMedicine 14:397-403, 2004

NPL 4: Yasuhiro Ogawa et al., Phase I study of a new radiosensitizercontaining hydrogen peroxide and sodium hyaluronate for topical tumorinjection: A new enzyme-targeting radiosensitization treatment, KochiOxydol-Radiation Therapy for Unresectable Carcinomas, Type II (KORTUCII), International Journal of Oncology 34: 609-618, 2009

NPL 5: Ryo Akama, Yasuhiro Ogawa, et al., Experimental Study onRadiation Sensitizing Effect of Intratumorally Administering HydrogenPeroxide to Mouse-transplanted Tumor, Usefulness of Adding HyaluronicAcid, Clinical Radiation Vol. 54, No. 12, 2009

NPL 6: Shibo Tokuhiro, Yasuhiro Ogawa, et al., Development of a novelenzyme-targeting radiosensitizer (KORTUC) containing hydrogen peroxidefor intratumoral injection for patients with low linear energytransfer-radioresistant neoplasms, Oncology Letters 1: 1025-1028, 2010

NPL 7: Yasuhiro Ogawa and et al., Safety and effectiveness of a newenzyme-targeting radiosensitization treatment (KORTUC II) forintratumoral injection for low-LET radioresistant tumors, InternationalJournal of Oncology 39: 533-560, 201

SUMMARY OF INVENTION Technical Problem

The radiation sensitizer or anti-cancer chemotherapy sensitizercomprising hydrogen peroxide, and hyaluronic acid or a salt thereof(hereinafter also referred to as “KORTUC”) that was previously developedby the present inventors must be locally injected into a tumor in thepatient under ultrasonographic guidance twice a week during radiotherapyor anti-cancer chemotherapy. Therefore, when the sensitizer is injected,the patient to be treated has a heavy physical and mental burden, suchas pain, and doctors who inject the sensitizer, as well as doctors orthe like who are in charge of ultrasonography, are also physicallyburdened and pressured by time. Even when KORTUC is used, the highestradiation sensitizing effect with the highest partial pressure of oxygenin tumors is obtained within 24 hours after injecting the sensitizer;therefore, irradiation with radiation or anti-cancer chemotherapy mustbe performed immediately after injecting the sensitizer.

In order to solve the above problem, an object of the present inventionis to provide a sensitizer that is for enhancing a therapeutic effect ofradiation or an anti-cancer agent on a tumor and that can prevent thedecomposition of hydrogen peroxide for a longer period of time andmaintains its effect.

Solution to Problem

The present inventors conducted extensive research to solve the aboveproblem. They found that when hydrogen peroxide is used together with ahydrogel comprising a crosslinked gelatin gel, hydrogen peroxideadministered into the body can be significantly inhibited fromdecomposition due to the function of antioxidants and can maintain theeffect of hydrogen peroxide administration for a longer period of timethan KORTUC. The inventors further found that even when the frequency ofhydrogen peroxide administration is reduced, using hydrogen peroxidetogether with a hydrogel comprising a crosslinked gelatin gel canprovide more potent radiation sensitizing effects and anti-cancerchemotherapy sensitizing effects than using KORTUC, due to the long-termsustained release of hydrogen peroxide immersed in the hydrogel.

The present invention has been accomplished based on the above findingsand includes the following.

I. Sensitizer for Enhancing Therapeutic Effect of Radiation orAnti-cancer Agent on Tumor

I-1. A sensitizer for enhancing a therapeutic affect of radiation or ananti-cancer agent on a tensor, the sensitizer comprising a combinationof:

-   -   (a) hydrogen peroxide and    -   (b) a hydrogel comprising a crosslinked gelatin gel, and the        sensitizer being locally administered to a tumor area.        I-2. The sensitizer according to Item I-1, wherein the tumor is        resistant to treatment, using radiation or an anti-cancer agent.        I-3. The sensitizer according to Item I-1 or I-2, wherein a        first administration of the sensitizer is performed during a        period from 3 days before a first irradiation with radiation or        a first administration of the anti-cancer agent until the start        of the irradiation or the administration of the anti-cancer        agent.        I-4. The sensitizer according to item I-3, wherein a second        administration of the sensitizer is performed 5 to 14 days after        the first administration of the sensitizer.        I-5. The sensitizer according to any one of Items I-1 to I-4,        wherein the crosslinked gelatin gel is prepared from a gelatin        having an isoelectric point of 4.5 to 5.5.        I-6. The sensitizer according to any one of Items I-1 to I-5,        wherein the crosslinked gelatin gel has a water content of 92 to        99.7%.        I-7. The sensitizer according to any one of Items I-1 to I-6,        which, in the form of a final formulation, is administered in an        amount of 0.1 to 10 mg, on a dry weight basis of the crosslinked        gelatin gel, per 200 mm³ of the tumor volume.        I-8. The sensitizer according to any one of Items I-1 to I-7,        which, in the form of the final formulation, contains hydrogen        peroxide in an amount of 0.01 to 3.5 wt. %.

II. Method For Treating Tumor

II-1. A method for treating a tumor, comprising the steps of:

-   -   (1) locally administering to a tumor area a sensitizer        comprising a combination of (a) hydrogen peroxide and (b) a        hydrogel comprising a crosslinked gelatin gel; and    -   (2) irradiating the tumor area with radiation or administering        an anti-cancer agent to a patient suffering from the tumor.        I-2. The method according to Item II-1, wherein the tumor is        resistant to treatment using radiation or an anti-cancer agent.        I-3. The method according to Item II-1 or II-2, wherein a first        administration of the sensitizer is performed during a period        from 3 days before a first irradiation with radiation or a first        administration of the anti-cancer agent until the start of the        irradiation or the administration of the anti-cancer agent.        II-4. The method according to Item II-3, wherein a second        administration of the sensitizer is performed 5 to 14 days after        the first administration of the sensitizer.        II-5. The method according to any one of Items II-1 to II-4,        wherein the crosslinked gelatin gel is prepared from a gelatin        having an isoelectric point of 4.5 to 5.5.        II-6. The method according to any one of Items II-1 to II-5,        wherein the crosslinked gelatin gel has a water content of 92 to        99.7%.        II-7. The method according to any one of Items II-1 to II-6,        wherein the sensitizer, in the form of a final formulation, is        administered in an amount of 0.1 to 10 mg, on a dry weight basis        of the crosslinked gelatin gel, per 200 mm³ of the tumor volume.        II-8. The method according to any one of Items II-1 to II-7,        wherein the sensitizer, in the form of the final formulation,        contains hydrogen peroxide in an amount of 0.01 to 3.5 wt. %.

III. Composition for Treating Tumor Using Radiation or Anti-Cancer Agent

III-1. A composition for treating a tumor using radiation or ananti-cancer agent, the composition comprising a combination of

-   -   (a) hydrogen peroxide and    -   (b) a hydrogel comprising a crosslinked gelatin gel, and the        composition being locally administered to a tumor area.        III-2. The composition according to Item III-1, wherein the        tumor is resistant to treatment using radiation or an        anti-cancer agent.        III-3. The composition according to Item III-1 or III-2, wherein        a first administration of the sensitizer is performed during a        period from 3 days before a first irradiation with radiation or        a first administration of the anti-cancer agent until the start        of the irradiation or the administration of the anti-cancer        agent.        III-4. The composition according to Item III-3, wherein a second        administration of the sensitizer is performed 5 to 14 days after        the first administration of the sensitizer. III-5. The        composition according to any of Items III-1 to III-4, wherein        the crosslinked gelatin gel is prepared from a gelatin having an        isoelectric point of 4.5 to 5.5.        III-6. The composition according to any of Items III-1 to III-5,        wherein the crosslinked gelatin gel has a water content of 92 to        99.7%.        III-7. The composition according to any of Items III-1 to III-6,        which, in the form of a final formulation, is administered in an        amount of 0.1 to 10 mg, on a dry weight basis of the crosslinked        gelatin gel, per 200 mm³ of the tumor volume.        III-8. The composition according to any of Items III-1 to III-7,        which, in the form of a final formulation, contains hydrogen        peroxide in an amount of 0.01 to 3.5 wt. %.        IV. Use in the Manufacture of Sensitizer for enhancing        Therapeutic Effect of Radiation or Anti-cancer Agent on Tumor        IV-1. Use of a composition for local administration to a tumor        area in the manufacture of a sensitizer for enhancing a        therapeutic effect of radiation or an anti-cancer agent on a        tumor, the composition comprising a combination of the following        components (a) and (b):    -   (a) hydrogen peroxide; and    -   (b) a hydrogel comprising a crosslinked gelatin gel.        IV-2. The use according to Item IV-1, wherein the tumor is        resistant to treatment using radiation or an anti-cancer agent.        IV-3. The use according to Item IV-1 or IV-2, wherein a first        administration of the sensitizer is performed during a period        from 3 days before a first irradiation with radiation or a first        administration of the anti-cancer agent until the start of the        irradiation or the administration of the anti-cancer agent.        IV-4. The use according to Item IV-3, wherein a second        administration of the sensitizer is performed 5 to 14 days after        the first administration of the sensitizer.        IV-5. The use according to any one of Items IV-1 to IV-4,        wherein the crosslinked gelatin gel is prepared from a gelatin        having an isoelectric point of 4.5 to 5.5.        IV-6. The use according to any of Items IV-1 to IV-5, wherein        the crosslinked gelatin gel has a water content of 92 to 99.7%.        IV-7. The use according to any of Items IV-1 to IV-6, wherein        the sensitizer, in the form of a final formulation, is        administered in an amount of 0.1 to 10 mg, on a dry weight basis        of the crosslinked gelatin gel, per 200 mm³ of the tumor volume.        IV-8. The use according to any of Items IV-1 to IV-7, wherein        the sensitizer, in the form of the final formulation, contains        hydrogen peroxide in an amount of 0.01 to 3.5 wt %.

Advantageous Effects of Invention

According to the present invention, sensitivity to the effects ofradiotherapy or anti-cancer chemotherapy can be increased even whenirradiation by radiation or administration of an anti-cancer agent isnot performed immediately after administration of the sensitizer.Further, the present invention can prolong tumor growth inhibitioneffects per dose of radiation for a long period of time, even comparedto KORTUC, and provide more potent tumor growth inhibition effects thanKORTUC.

Furthermore, while KORIOC must be injected into an affected area twice aweek, to provide effects, the sensitizer according to the presentinvention is effective with administration once a week at most, thusreducing physical, mental, and time burden on the person to be treated.

Furthermore, the present invention can provide more potent tumor growthinhibition effects and reduce the risk of tumor recurrence, evencompared to KORTUC.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a bolus and a device to which each mouse was secured whenthe mouse was irradiated with radiation.

FIG. 2 shows the survival curve of each group after irradiation withradiation (⋄: control group, ▪: KORTUC-administered group, Δ:MedGel-administered group). Specifically, after tumor cells wereimplanted in mice, PBS+hydrogen peroxide (control), hyaluronicacid+hydrogen peroxide (KORTUC), or MedGel+hydrogen peroxide (MedGel)were administered to the mice, and 72 hours after the administration,the mice were irradiated with radiation.

FIG. 3 shows the tumor volume curve of each group after irradiation withradiation (⋄: control group, ▪: KORTUC-administered group, Δ;MedGel-administered group.) Specifically, after turner cells wereimplanted in mice, PBS+hydrogen peroxide (control), hyaluronicacid+hydrogen peroxide (KORTUC), or MedGel+hydrogen peroxide (MedGel)were administered to the mice, and 72 hours after the administration,the mice were irradiated with radiation.

FIG. 4 shows a comparison in tumor volume on day 30 from the start ofmeasurement in the graphs shown in FIG. 3 (from left to right, thecontrol group, KORTUC-administered group, and MedGel-administeredgroup).

FIG. 5 shows time-dependent changes of MedGel particles in oxydol duringthe period up to 10 days later.

FIG. 6 shows the effects on tumor volume of irradiation with radiationperformed 48 hours after the administration of each sensitizer to atumor. The tumor volume was measured on day 37 after the administrationof each sensitizer (on day 35 after irradiation with radiation). Thecontrol refers to a group that was only irradiated with radiation.KORTUC refers to a group to which KOURTUC was administered and that wasirradiated with radiation. PI5-100 refers to a group to which 100 μl ofthe sensitizer obtained in Formulation Example 1 was administered andthat was irradiated with radiation. PI5-30 refers to a group to which 30μl of the sensitizer obtained in Formulation Example 1 was administeredand that was irradiated with radiation. PI9-2 refers to a group to whichthe sensitizer obtained in Formulation Example 2 was administered in anamount of 2 mg, on a dry weight basis of PI-9, and that was irradiatedwith radiation. PI9-0.6 refers to a group to which the sensitizerobtained in Formulation Example 2 was administered in an amount of 0.6mg, on a dry weight basis of PI-9, and that was irradiated withradiation.

FIG. 7 shows the effect on tumor volume of irradiation with radiationperformed 72 hours after the administration of each sensitizer to atumor. The tumor volume was measured on day 37 after the administrationof the sensitizer (on day 34 after irradiation with radiation). Thecontrol refers to a group that was only irradiated with radiation.KORTUC refers to a group to which KOURTUC was administered and that wasirradiated with radiation. PI5-30 refers to a group to which 30 μl ofthe sensitizer in Formulation Example 1 was administered and that wasirradiated with radiation. PI9-0.6 refers to a group to which thesensitizer of Formulation Example 2 was administered in an amount of 0.6mg, on a dry weight basis of PI-9, and that was irradiated withradiation.

DESCRIPTION OF EMBODIMENTS I. Sensitizer For Enhancing TherapeuticEffect of Radiation or Anti-Cancer Agent on Tumor I-1. Composition ofSensitizer

A feature of the sensitizer for enhancing a therapeutic effect ofradiation or an anti-cancer agent on a tumor according to the presentinvention (sometimes simply referred to as the “sensitizer” in thisspecification) is that the sensitizer comprises (a) hydrogen peroxideand (b) a hydrogel comprising a crosslinked gelatin gel.

In the present invention, “the sensitizer for enhancing a therapeuticeffect of radiation or an anti-cancer agent” refers to a pharmaceuticalcomposition that can be locally administered to a tumor area before,simultaneously with, or after irradiation with radiation in radiotherapy(irradiation with radiation) or administration of an anti-cancer agentin anti-cancer chemotherapy (administration of an anti-cancer agent),and that can increase the effect of radiotherapy or anti-cancerchemotherapy on the tumor area.

In the present invention, “tumor” refers to tumors in general,regardless of whether benign or malignant, or epithelial ornon-epithelial. Malignant tumors are preferable. Although the tumor isnot particularly limited in sensitivity to therapy using radiationand/or an anti-cancer agent, tumors that are resistant to therapy usingradiation or an anti-cancer agent are preferable.

In the present invention, “tumor area” refers to a tumor tissue in whichtumor cells are mainly present. The tumor tissue includes tissue inwhich tumor cells and normal cells and/or normal tissue are presenttogether. When tumor cells and normal cells and/or normal tissue arepresent together, the volume or number of tumor cells relative to thatof normal cells and/or normal tissue is not particularly limited.

Therapeutic effects can be evaluated by using general evaluationmethods, such as the percentage of tumor tissue shrinkage, patientsurvival rate, and recurrence rate after treatment.

The effect of linear-accelerator-based radiotherapy on tumor cellsdepends about 70% on the production of active oxygen species, such ashydroxyl radicals. The mechanisms of various anti-cancer agents(carcinostatic agents) also have this point in common. Therefore, asubstance that increases sensitivity to radiotherapy effects alsoincreases sensitivity to anti-cancer chemotherapy effects on tumors atthe same time.

“Hydrogen peroxide” as used in the present invention refers to hydrogenperoxide molecules (H₂O₂; molecular weight of 34) unless otherwisespecified.

“Aqueous hydrogen peroxide solution” refers to hydrogen peroxidedissolved in distilled water in the Japanese Pharmacopoeia or the likeunless otherwise specified. The concentration of hydrogen peroxide inthe hydrogen peroxide solution is 0.1 to 30 w/v %, and preferably 2.5 to30 w/v %.

“Oxydol” as used in this specification refers to oxydol in accordancewith the Japanese Pharmacopeia and is an aqueous hydrogen peroxidesolution containing 2.5 to 3.5 w/v % of hydrogen peroxide.

In this specification, w/v % refers to weight/volume percentconcentration unless otherwise specified.

Gelatin that is a starting material for the “crosslinked gelatin gel”used in the present invention can be obtained by denaturing collagenthat can be obtained from various body parts, such as skin, bones,tendons, ligaments, fins, and tails, of various kinds of animal species,such as cows, pigs, and fish, by various treatments such as alkalinehydrolysis, acid hydrolysis, and/or enzymatic decomposition. The gelatinis preferably a bovine- or porcine-derived gelatin, and particularlypreferably bovine-derived gelatin.

The gelatin may be naturally occurring collagen or one obtained bydenaturing collagen produced by genetic recombination technology usingthe following method.

The method for obtaining gelatin is not particularly limited. Gelatinmay be extracted from bones, etc., of the animals mentioned above, orcommercially available products such as those obtained from NittaGelatin Inc., Nippi Inc., and Gunze Ltd. may be used. Gelatin that doesnot contain any endotoxin is preferable.

The gelatin may be, for example, alkali-treated gelatin (acidic gelatin)with an isoelectric point of 4.5 to 5.5, and preferably 4.8 to 5.0; oracid-treated gelatin (alkaline gelatin) with an isoelectric point of 7.0to 10.0, and preferably 8.0 to 9.0. Acidic gelatin with an isoelectricpoint of 4.8 to 5.0 is preferable.

Acidic gelatin can be obtained by treating collagen using lime or thelike according to a usual method for about 2 to 3 months. Thedeamidation rate after alkali treatment is about 98 to 100%.

Alkaline gelatin can be obtained by treating collagen using an inorganicacid such as hydrochloric acid or sulfuric acid for about several tensof hours to several days. The deamidation rate after acid treatment isabout 0 to 2%.

Not only one kind of gelatin, but also gelatins with different materialsand different physical properties, such as solubility, molecular weight,and isoelectric point, may be used in appropriate blends.

Although the properties of the gelatin vary according to the materialand treatment method used, gelatin having any property can be used as astarting material for the crosslinked gelatin gel of the presentinvention as long as the gelatin does not include a pharmaceuticallynon-acceptable component.

The “hydrogel comprising a crosslinked gelatin gel” used in the presentinvention contains as a main component a gelatin gel formed bycrosslinking gelatin with a chemical crosslinking agent or by heattreatment, ultraviolet irradiation, or electron beam irradiation.

The hydrogel comprising a crosslinked gelatin gel may contain, inaddition to a crosslinked gelatin gel, dyes, proteins other thancollagen, and/or salts, and like impurities in an amount within apharmaceutically acceptable range.

The hydrogel comprising a crosslinked gelatin gel may contain thecrosslinked gelatin gel in an amount of, for example, 95 to 100 w/v %,more preferably 98 to 100 w/v %, and most preferably 99.5 to 100 w/v %.

The chemical crosslinking agent for crosslinking the gelatin usable inthe preparation of a crosslinked gelatin gel in the present invention isnot particularly limited as long as it is not toxic to living bodies.For example, glutaraldehyde, water-soluble carbodiimides such as1-ethyl-3- (3-dimethylaminopropyl)carbodiimide hydrochloride and1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-metho-p-toluenesulfonate,bisepoxy compounds, formalin, and the like are preferable.Glutaraldehyde and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride are particularly preferable.

These crosslinking treatments may be used in combination. Alternatively,a crosslinked hydrogel may be prepared by physical cross-linking usingsalt bridge, electrostatic interaction, hydrogen bond, hydrophobicinteraction, or the like. Using a chemical crosslinking agent ispreferable from the viewpoint of the strength of crosslinking.

The degree of gelatin crosslinking can be suitably selected according tothe rate (number of days) of decomposition of the crosslinked gelatingel in the living body. The decomposition rate of the crosslinkedgelatin gel can be measured, for example, by using the following method.60 μl of PBS is added dropwise per mg of the crosslinked gelatin gel, ona dry weight basis, and allowed to stand at 37° C. for 1 hour or at 4°C. for 12 hours to 24 hours. After measuring the total weight of thecrosslinked gelatin gel impregnated with PBS, the crosslinked gelatingel is implanted subcutaneously into the back of several mice. Some ofthe mice are slaughtered every day or every 2 to 3 days to remove theimplanted crosslinked gelatin gel and the wet weight of the crosslinkedgelatin gel is measured. The point of time when the weight of theremoved crosslinked gelatin gel has become approximately half of the wetweight of the PBS-impregnated crosslinked gelatin measured beforeimplantation is defined as the half-life of the crosslinked gelatin gel(unit: number of days).

The crosslinked gelatin gel used in the present invention has ahalf-life of 5 to 25 days, preferably 8 to 22 days, more preferably 10to 20 days, and even more preferably 14 to 19 days.

The degree of crosslinking of the crosslinked gelatin can be evaluatedas water content. The water content can be determined according to, forexample, the method disclosed in the report of Tabata, Y et al. (Tabata,Y et al., J. Controlled Release, 31; 189-199, 1994, Tabata, Y et al.,Tissue Eng. 1999 (2): 127-38). For example, 1 mg of a dry crosslinkedgelatin gel is immersed in 1 to 3 ml of PBS (pH of 7.4) and incubated at37° C. for about 24 hours with occasional agitation. After removingexcess PBS, the wet weight of swollen crosslinked gelatin gel afterimpregnation with PBS (wet weight of the crosslinked gelatin gel afterswelling) is measured.

The weight of absorbed water is calculated by subtracting the weight ofthe dry crosslinked gelating gel from the wet weight of the crosslinkedgelatin gel after swelling. Specifically, the weight of absorbed wateris calculated by the following formula.

Weight of absorbed water=(Wet weight of the crosslinked gelatin gelafter swelling)−(Weight of the dry crosslinked gelatin gel)

The water content is calculated by dividing the weight of absorbed waterby the wet weight of the crosslinked gelatin gel after swelling andmultiplying the value by 100. Specifically, the water content iscalculated by the following formula.

Water content (%)=(Weight of absorbed water/Wet weight of thecrosslinked gelatin gel after swelling)×100

The crosslinked gelatin gel used in the present invention has a watercontent of 92 to 99.7%, preferably 93 to 98.5%, and more preferably 95to 98%. The lower the water content of the crosslinked gelatin gel, thelower the biodecomposition rate of the crosslinked gelatin. Thehalf-life of the crosslinked gelatin gel with a water content of 98.8%is about 6 to 8 days. The half-life of the crosslinked gelatin gel witha water content of 97.6% is about 10 to 14 days. The half-life of thecrosslinked gelatin gel with a water content of 96.9% is about 16 to 20days.

A crosslinked gelatin gel having the above water content can beobtained, for example, by adjusting the concentrations of the gelatinand the crosslinking agent to the following ranges when the crosslinkedgelatin gel is produced: a gelatin concentration of 1 to 20 wt. % and acrosslinking agent concentration of 0.01 to 1 wt. %. Further, acrosslinking agent with a desired water content can be obtained, forexample, by appropriately adjusting the crosslinking reaction conditionsto a temperature of 0 to 40° C. and a reaction time of 1 to 48 hours.

For example, when glutaraldehyde is used as a crosslinking agent, ifglutaraldehyde is added to a 3 to 5 wt. % aqueous gelatin solution to afinal concentration of 0.8 to 1.0 wt. %, and a crosslinking agent isperformed at 4° C. for about 12 hours, a crosslinked gelatin gel with awater content of approximately 94% can be obtained. If glutaraldehyde isadded to a 3 to 5 wt. % aqueous gelatin solution to a finalconcentration of 0.6 to 0.7 wt. % and a crosslinking reaction isperformed under the same conditions as above, a crosslinked gelatin gelwith a water content of approximately 97% can be obtained. Ifglutaraldehyde is added to a 3 to 5 wt. % aqueous gelatin solution to afinal concentration of 0.1 to 0.4 wt. % and a crosslinking reaction isperformed, a crosslinked gelatin gel with a water content ofapproximately 98% can be obtained.

When 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)is used as a crosslinking agent, if EDC is appropriately added to a 1 to10 wt. % aqueous gelatin solution to a final concentration of 0.01 to 10wt. % and a reaction is allowed to proceed at 4° C. for 24 hours, acrosslinked gelatin gel with a water content of 92 to 99.7% can beobtained.

Gelatin becomes insoluble in water when crosslinked. The term“water-insoluble” refers to the property of being able to be impregnatedwith a solution containing water and swelling but not dissolving in anaqueous solution containing water. The solubility of the crosslinkedgelatin gel in water can be determined, for example, by adding 1 g of adry powder crosslinking gelatin per 100 g of water at 60° C. anddetermining the gelatin gel to be water-soluble if the amount of gelatindissolved after being allowed to stand for 1 hour is 0.1 g or more, anddetermining the gel to be water-insoluble if the amount of gelatindissolved after being allowed to stand for 1 hour is less than 0.1 g.

The form of the crosslinked gelatin gel used in the present invention isnot particularly limited. The crosslinked gelatin gel may be, forexample, in the form of a cylinder, prism, sheet, disk, sphere,particle, granule, powder, paste, etc. When the sensitizer of thepresent invention is implanted into a tumor area, the crosslinkedgelatin gel in the form of a cylinder, prism, sheet, or disk ispreferable. The gel in the form of a sheet or disk is more preferable.When the sensitizer of the present invention is used in the form offormulation that is directly injected into a tumor area orintravascularly injected into a tumor area, the crosslinked gelatin gelin the form of a sphere, particle, granule, powder, or paste ispreferable.

The crosslinked gelatin gel can be formed, for example, according to themethods disclosed in JP2004-203829A and JP2005-325075A.

Specifically, the crosslinked gelatin gel in the form of a cylinder, aprism, a sheet, or a disk can be prepared by mixing an aqueous gelatinsolution with an aqueous solution of a crosslinking agent, then castingthe mixture into a mold having a desired shape, and allowing acrosslinking reaction to proceed. The formed gelatin gel may be used asit is, or an aqueous solution of a crosslinking agent may be added afterdrying. To quench the crosslinking reaction, the reaction product isbrought into contact with a low-molecular-weight substance having anamino group, such as ethanolamine or glycine, or an aqueous solutionhaving a pH of 2.5 or less is added. The obtained crosslinked gelatingel is washed with distilled water, ethanol, 2-propanol, acetone, or thelike, and used to prepare a formulation.

The crosslinked gelatin gel in the form of a paste can be prepared byusing a method similar to the method of preparing a crosslinked gelatingel in the form of a cylinder, prisma, sheet, or disk.

The crosslinked gelatin gel in the form of a sphere, particle, orgranule, can be obtained, for example, by the following method. Anaqueous gelatin solution is placed in a device formed by attaching astirring motor (e.g., Three-One Motor, manufactured by Shinto ScientificCo,, Ltd., or EYELA mini D.C. Stirrer) and a stirring propeller ofTeflon (trademark) to a three-necked round flask and fixing the flaskequipped with the stirring motor and the stirring propeller. After anoil such as olive oil is added and the resulting mixture is stirred atabout 200 to 600 rpm to give a W/O emulsion, an aqueous solution of acrosslinking agent is added. Alternatively, after an aqueous gelatinsolution pre-emulsified in olive oil (e.g., Advantec TME-21 vortex mixeror PT10-35 homogenizer polytron) is added dropwise to olive oil to givea fine particulate W/O emulsion, an aqueous solution of a crosslinkingagent is added thereto. After a crosslinking reaction is allowed toproceed, the crosslinked gelatin gel is recovered by centrifugation,washed with acetone, ethyl acetate, etc., further washed with IPA,ethanol, etc., and dried. Subsequently, 100 mM glycine is added to anaqueous solution containing Tween 80, and the particles are suspended inthe solution to quench the crosslinking reaction. The obtainedcrosslinked gelatin gel particles are washed successively with IPA,distilled water containing Tween 80, distilled water, etc., and used toprepare the formulation.

When the crosslinked gelatin gel particles are coagulated, for example,ultrasonicaticn (preferably within about 1 minute under cooling) and thelike may be applied.

By pre-emulsifying, crosslinked gelatin gel in the form of fineparticles having an average particle size of 20 μm or less can beobtained.

The average particle size of the obtained crosslinked gelatin gelparticles is 1 to 1,000 μm. Particles having a necessary size areappropriately sieved according to the object, and used. For localadministration, particles having an average particle size of 10 to 150μm are preferable. As used herein, the average particle size means anaverage diameter of the particles, which is assumed by passing thesieves. The water content of the obtained crosslinked gelatin gelparticles is about 50 to 99 w/w %, and particles having a preferablewater content can be

appropriately prepared.

The crosslinked gelatin gel thus obtained can also be vacuum-dried orfreeze-dried.

For freeze drying, for example, a crosslinked gelatin gel is placed, indistilled water, frozen in liquid nitrogen for 30 minutes or more at−80° C. for at least 1 hour and then dried in a freeze dryer for 1 to 3days.

The dry crosslinked gelatin gel may be a commercially available product,such as MetJel (registered trademark) manufactured by MedGelCorporation.

The sensitizer of the present invention is a composition comprising acombination of hydrogen peroxide (herein also referred to as “component(a)”) and a hydrogel comprising a crosslinked gelatin gel (herein alsoreferred to as “component (b)”).

The phrase “comprising a combination” as used herein inclusively meansall the following cases for the sensitizer of the present invention:

-   -   (i) the sensitizer contains both component (a) and component (b)        from the beginning (a combination drug), i.e., component (b) is        impregnated with component (a), or (i)′ component (b) is        impregnated with component (a) and the resulting product is        floated or suspended in physiological saline, etc.;    -   (ii) a formulation containing component (a) and a formulation        containing component (b) are separately packaged and sold as a        combination (a kit); or    -   (iii) a formulation containing component (a) and a formulation        containing component (b) are separately packaged and are in        separate market distribution channels, and are combined when        used. That is, “the sensitizer comprising a combination” as used        herein means that the sensitizer that is ultimately used (herein        also referred to as “final formulation”) contains both        component (a) and component (b), and the form during the        distribution stage, including sale, is not particularly limited.

The phrase “comprising a combination of component (a) and component (b)”inclusively means “containing component (a) and component (b),”“substantially consisting of component (a) and component Ob),” and“consisting of component (a) and component (b).”

The proportion of hydrogen peroxide contained as component (a) in thesensitizer of the present invention is not limited, and can be suitablyselected from the range that the amount of hydrogen peroxide in thefinal formulation is in the range of 0.01 to 3.5 w/v %. The amount ispreferably in the range of 0.1 to 3 w/v %, and more preferably 0.5 to 2w/v %.

The proportion of the hydrogel comprising a crosslinked gelatin gen tohydrogen peroxide is not particularly limited, and can be suitablyselected from the range that the amount of the hydrogel comprising acrosslinked gelatin gel per 300 μg of hydrogen peroxide (10 μl of a 3w/v % aqueous hydrogen peroxide solution) is in the range of 0.1 to 1.00mg (dry weight), preferably 0.5 to 50 mg (dry weight), and morepreferably 0.8 to 10 mg (dry weight).

The proportion of the hydrogel comprising a crosslinked gelatin gel inthe sensitizer (final formulation) of the present invention is notparticularly limited, and can be suitably selected from the range of0.05 to 20 w/v %, preferably 0.1% to 10 w/v %, and more preferably 0.5to 5 w/ %. The weight (w) of the hydrogel comprising a crosslinkedgelatin gel is on a dry weight basis.

The method for impregnating the hydrogel comprising a crosslinkedgelatin gel with an aqueous hydrogen peroxide solution comprisesweighing a required amount of the hydrogel comprising a dry crosslinkedgelatin gel in the form of a cylinder, prism, sheet, disk, sphere,particle, granule, or powder, and adding the aqueous hydrogen peroxidesolution dropwise to the hydrogel comprising a crosslinked gelatin gelusing a pipette, etc, in an amount to achieve the proportion mentionedabove, followed by incubation, for example, at 30 to 37° C. for about 15minutes to about 2 hours, or at 2 to 8° C. for about 12 to 24 hours, andpreferably at 37° C. for about 1 hour, or at 4° C. for about 12 to 18hours.

To fix the qualities as a sensitizer, the aqueous hydrogen peroxidesolution is preferably added dropwise in a volume such that thesensitizer can be impregnated with all the aqueous hydrogen peroxidesolution added dropwise.

The product obtained by impregnating the hydrogel comprising acrosslinked gelatin gel with, the aqueous hydrogen peroxide solution byusing the above method may be used as a sensitizer as is or after beingfurther floated or suspended in pharmaceutically acceptablephysiological saline or phosphate buffer (such as sodium chloride,disodium hydrogen phosphate, and sodium dihydrogen phosphate). Even whenthe hydrogel comprising a crosslinked gelatin gel is impregnated with anaqueous hydrogen peroxide solution and further floated or suspended inphysiological saline or the like, the final contents of the hydrogenperoxide and the hydrogel comprising a crosslinked gelatin gel can beadjusted to the ranges mentioned above.

The liquid properties of the sensitizer of the invention are notparticularly limited as long as it is compatible with the human body.The sensitizer is preferably adjusted to a pH of 6 to 8.5, and morepreferably a pH of 6.5 to 8.0.

I-2. Method for Applying the Sensitizer

The sensitizer of the present invention is in the form of a liquid(including a solution, an emulsion, and a suspension), a gel, or anelastic solid. As long as the sensitizer is in such a form, the mode ofuse (usage) is not particularly limited. For example, when thesensitizer of the present invention is a liquid or gel externalformulation, the sensitizer can be locally administered to the tumorarea, for example, by the following methods: methods of directlyspraying, applying, or attaching the sensitizer onto the tumor areaduring the irradiation with radiation in the radiotherapy or duringadministration of an anti-cancer agent in anti-cancer chemotherapy, andmethods of impregnating sterile cotton or gauze with the sensitizer andapplying the impregnated sterile cotton or gauze to the tumor area to beirradiated with radiation during the irradiation.

When the sensitizer of the present invention is a liquid or gelinjection, the injection can be directly injected into the tumor area tobe treated, preferably intratumorally injected, using a syringe or thelike, or indirectly injected via an angiography catheter into the tumorarea, before, simultaneously with, or after the irradiation withradiation or the administration of an anti-cancer agent.

Specifically, the sensitizer is preferably injected by using a syringewith a needle of about 21 gauge while observing the state of permeationof the sensitizer into the tissue under ultrasonographic guidance. Inthis case, the sensitizer can be distributed widely to the tissue underultrasonographic guidance by modifying the depth and direction of theinjection needle. The sensitizer may be injected into the tumor area viathe vasa vasorum of the tumor. The dose of the sensitizer in the presentinvention administered to the tumor area varies depending on the size ofthe tumor and the administration method. For example, when thesensitizer is injected into the tumor area, the dose

(injection amount) may be typically about 1 to 5 ml, and preferablyabout 3 ml.

When the sensitizer of the present invention is in the form of anelastic solid, the sensitizer may be implanted by incising the tumorarea or the area after resection of the tumor using a scalpel or thelike.

In another embodiment, the dose of the sensitizer in the presentinvention can also be set according to the tumor volume. The tumorvolume (V) can be calculated, for example, by measuring the minor axis(W) and major axis (L) of the tumor using a Vernier caliper, andsubstituting the obtained values into the following equation:V=(W²×L)/2. The lower limit of the dose of the sensitizer administeredto mice in accordance with the tumor volume is 0.1, 0.2, 0.3, 0.4, or0.5 mg, on a dry weight basis of the crosslinked gelatin gel containedin the final formulation, per 200 mm3 of the tumor volume. The upperlimit of the dose of the sensitizer is 10, 5, 3, 2, or 1 mg, on a dryweight basis of the crosslinked gelatin gel contained in the finalformulation, per 200 mm³ of the tumor volume. Any combination of theupper limit and lower limit may be used. The lower limit is preferably0.1 mg, and the upper limit is preferably 1 mg.

The dose administered to humans can be set in accordance with the doseadministered to mice. If necessary, the sensitizer may be administeredto humans at 5 to 10 times the dose administered to mice. The lowerlimit of the dose of the sensitizer administered to a human inaccordance with the tumor volume may be, for example, 0.5, 1, 1.5, 2, or2.5 mg, on a dry weight basis of the crosslinked gelatin gel containedin the final formulation, per 200 mm³ of the tumor volume. The upperlimit of the dose of the sensitizer may be, for example, 5, 8, 10, 15,or 20 mg, on a dry weight basis of the crosslinked gelatin gel containedin the final formulation, per 200 mm³ of the tumor volume. Anycombination of the upper limit and lower limit may be used. The lowerlimit is preferably 0.5 mg, and the upper limit is preferably 5 or 10mg.

The sensitizer of the present invention is preferably in the form of aninjection (a radiotherapy sensitizer injection or anti-cancerchemotherapy sensitizer injection). The injection can be obtained bypreparing an aqueous solution using water for injection (distilled waterfor injection, sterile water for injection, etc.), an isotonizing agent,a pH control agent, a buffer solution, and the like, adding component(a) and component (b) to the resulting aqueous solution in amounts toachieve the proportions mentioned above, placing the resulting mixturein a container and sealing the container, followed by sterilizationusing high-pressure steam sterilization, hot water immersionsterilization, or the like. Alternatively, the injection (a preparationprepared just before use) can be obtained by mixing a formulationcontaining component (a) and a formulation containing component (b) atthe time of use, optionally using water for injection (distilled waferfor injection, sterile water for injection, etc.).

Examples of usable isotonizing agents include sodium chloride, glycerin,glucose, polyethylene glycol, propylene glycol, D-mannitol, fructose,xylitol, sodium dihydrogen phosphate, and sodium phosphate. Sodiumchloride is preferably used. Examples of pH control agents includehydrochloric acid and sodium .hydroxide. As described above, theinjection is adjusted to a pH range of 6 to 8.5, and preferably to a pHrange of 6.8 to 7.8. Examples of buffers that can be used formaintaining the pH include phosphate buffers, Tris buffers, and acetatebuffers. A phosphate buffer is preferably used.

I-3. Radiotherapy Using the Sensitizer

As described above, in radiotherapy, the sensitizer of she invention isapplied to a tumor area to be irradiated with radiation before theirradiation. The sensitizer can be preferably used in radiotherapy oftumors that are resistant to radiotherapy.

Examples of tumors that are radioresistant include tumors that have manyhypoxic tumor cells and tumors that have large amounts of antioxidativeenzymes. The biggest problem with linear-accelerator-based cancerradiotherapy, which is commonly used currently, is the presence ofradioresistant cancer cells. The radioresistant tumor tissues are mostlyin hypoxic regions and are resistant to radiotherapy. The radiationresistance of these cells is considered to be attributable to thefollowing: in a hypoxic state, DNA damage, etc., of the cells induced byradiation are not fixed by oxygen; furthermore, reactive oxygen speciesproduced in tumor cells by radiation are eliminated by anti-oxidativeenzymes, thus making it difficult to induce apoptosis. Specific examplesof radioresistant tumors include malignant melanomas, malignantglioblastomas, and various types of sarcomas such as osteosarcomas, aswell as nearly all types of locally advanced cancers that have grown toseveral centimeters or more.

Radiotherapy using the sensitizer of the invention can be performed byfirst applying the sensitizer to the area of the tumor to be irradiatedwith radiation, and then irradiating the affected part with X-rays orelectron beams preferably using a linear accelerator. The x-rayirradiation conditions differ depending on how far the tumor hasadvanced, its size, and the like. One dose of radiation may typically be1.5 to 3 Gy, preferably about 2 Gy, and the irradiation may be performed2 to 5 times a week, and more preferably about 4 or 5 times a week, overa period of 1 to 5 weeks, with a total dose of 20 to 70 Gy, preferablyabout 40 to 70 Gy, and more preferably about 50 to 60 Gy. The electronbeam conditions also differ depending on how far one tumor has advanced,its size, and the like. One dose of radiation may typically 2 to 5 Gy,preferably about 4 Gy, and the irradiation may be performed 1 to 5 timesa week, and more preferably about 2 or 3 times a week, over a period of1 to 5 weeks, to a total dose of 30 to 70 Gy, and preferably about 40 to60 Gy.

The sensitizer of the invention may be administered simultaneously withor before the first irradiation with radiation. More specifically, thefirst administration of the sensitizer of the present invention ispreferably performed during the period from 3 days before the start ofthe irradiation with radiation until the start of the irradiation, morepreferably during the period from 2 days before the start of theirradiation with radiation until the start of the irradiation, and evenmore preferably 1 day before the start of the irradiation until thestart of the irradiation, or immediately before the irradiation withradiation. The second or later administration of the sensitizer can beperformed 5 to 14 days after the previous administration of thesensitizer, and more preferably 7 to 10 days after the previousadministration.

For example, when the irradiation with radiation is performed 4 to 5times a week for 4 weeks, if the irradiation is stated on Monday, thesensitizer of the present invention may be administered before theirradiation every Monday.

The frequency of administration of the sensitizer of the presentinvention can be increased or decreased according to the therapeuticeffects on tumors.

When radiotherapy is performed in advance and the effect of irradiationwith radiation is not obtained, use of the sensitizer of the presentinvention may be started after the start of radiotherapy.

When radiotherapy is started after a surgical operation, radiotherapymay be started in advance and use of the sensitizer of the presentinvention may be started, for example, 5 to 14 days, and more preferably6 to 8 days, after the start of irradiation with radiation.

For example, when radiotherapy is performed after breast conservativesurgery, the whole breast is irradiated with a dose of 50 Gy (2Gy×5times/(week)). When tumor cells are observed at the surgical marginduring the surgery, for example, the tumor cells can be boost-irradiatedwith an electron beam to a total dose of 10 Gy (2 Gy×5 times). Inanother embodiment, for example, the whole breast is irradiated to 44 Gy(2.75 Gy×16 times), and boost-irradiated to 9 Gy (3 Gy×3 times). Thesensitizer of the present invention can be locally administered to thetumor area, for example, 1 week after the start of the irradiation. Thefrequency of administration of the sensitizer may be, for example, 1 to4 times.

I-4. Anti-Cancer Chemotherapy Using the Sensitizer

The sensitizer of the invention may be used on the tumor area to betreated, before therapy with an anti-cancer agent. The sensitizer can bepreferably used on tumors that are resistant to therapy with ananti-cancer agent or that are relatively large. For example, many solidtumors, such as stomach cancer, non-small-cell lung cancer, colorectaland rectal cancer, liver cancer, pancreatic cancer, uterine cancer, andesophageal cancer, are resistant to anti-cancer agents. With respect tosuch solid tumors, nearly all locally advanced solid tumors tend to beresistant to anti-cancer agents.

The sensitizer of the present invention can be administeredsimultaneously with, or before the first administration of theanti-cancer agent. More specifically, the first administration of thesensitizer of the present invention is preferably performed during theperiod from 3 days before the start of the administration of theanti-cancer agent until the start of the administration, more preferablyduring the period from 2 days before the start of the administration ofthe anti-cancer agent until the start of the administration, and evenmore preferably 1 day before the start of the administration of theanti-cancer agent until the start of the administration, or immediatelybefore the start of the administration of the anti-cancer agent. Thesecond or later administration of the sensitizer can be performed 5 to14 days after the previous administration of the sensitizer, and morepreferably 7 to 10 days after the previous administration.

For example, when the anti-cancer agent is administered once to fivetimes per week for 4 weeks, and if the administration of the anti-canceragent is started on Monday, the sensitizer of the invention may beadministered before the start of the administration of the anti-canceragent every Monday.

The frequency of administration of the sensitizer of the presentinvention can be increased or decreased according to the therapeuticeffects on tumors.

When an anti-cancer agent is administered in advance and the effect ofthe anti-cancer agent is not obtained, use of the sensitizer of thepresent invention may be started after the start of the administrationof the anti-cancer agent.

Examples of anti-cancer agents (carcinostatic agents) used in thepresent invention include alkylating agents such as cyclophosphamide,ifosfamide, busulfan, melphalan, bendamustine hydrochloride, nimustinehydrochloride, ranimustine, dacarbazine, procarbazine hydrochloride, andtemozolomide; antimetabolites such as methotrexate, pemetrexed sodium,fluorouracil, doxifluridine, capecitabine, tegafur, cytarabine,cytarabine ocfosfate hydrate, enocitabine, gemcitabine hydrochloride,mercaptopurine hydrate, fludarabine phosphate, nelarabine, pentostatin,cladribine, levofolinate calcium, calcium folinate, hydroxycarbamide,L-asparaginase, and azacitidine; antitumor antibiotics such asdoxorubicin hydrochloride, daunorubicin hydrochloride, pirarubicin,epirubicin hydrochloride, idarubicin hydrochloride, aclarubicinhydrochloride, amrubicin hydrochloride, mitoxantrone hydrochloride,mitomycin C, actinomycin D, bleomycin, peplomycin sulfate, andzinostatin stimalamer; microtubule inhibitors such as vincristinesulfate, vinblastine sulfate, vindesine sulfate, vinorelbine tartrate,paclitaxel, docetaxel hydrate, and eribulin mesylate; hormonal agentssuch as anastrozole, exemestane, letrozole, tamoxifen citrate,toremifene citrate, fulvestrant, flutamide, bicalutamide,medroxyprogesterone acetate, estramustine phosphate sodium hydrate, andleuprolide acetate; platinum drugs such as cisplatin, miriplatinhydrate, carboplatin, nedaplatin, and oxaliplasin; topoisomerase Iinhibitors such as irinotecan hydrochloride hydrate and nogitecanhydrochloride; topoisomerase II inhibitors such as etoposide andsobuzoxane; cytokines such as interferon γ1a, teceleukin, andcelmoleukin; antibody drugs such as trastusumab, rituximab, gemtuzumabozogamicine, bevacizumab, and cetuximab; radioimmunotherapeutic agentssuch as ibritumomab tiuxetan; molecular target drugs such as gefitinib,imatinib mesylate, bortezomib, erlotinib hydrochloride, sorafenibtosylate, sunitinib malate, thalidomide, nilotinib hydrochloridehydrate, dasatinib hydrate, lapatinib tosylate hydrate, everolimus,lenalidomide hydrate, dexamethasone, temsirolimus, vorinostat,tretinoin, and tamibarotene; non-specific immune stimulants such asOK-432, dry BCG, Coriolus versicolor polysaccharide formulation,lentinan, and ubenimex; and anti-cancer agents such as aceglatone,porfimer sodium, talaporfin sodium, ethanol, and arsenic trioxide.

Examples of more preferable anti-cancer agents include anthracyclineanti-cancer agents such as doxorubicin hydrochloride, daunorubicinhydrochloride, pirarubicin, epirubicin hydrochloride, idarubicinhydrochloride, aclarubicin hydrochloride, amrubicin hydrochloride, andmitoxantrone hydrochloride; platinum anti-cancer agents such ascisplatin, miriplatin hydrate, carboplatin, nedaplatin, and oxaliplatin;and pyrimidine antimetabolite-based anti-cancer agents such asfluorouracil, doxifluridine, capecitabine, tegafur, cytarabine,cytarabine ocfostate hydrate, enocitabine, and gemcitabinehydrochloride.

These anti-cancer agents or carcinostatic agents can be used singly orin a combination of two or more.

The method of administering the anti-cancer agent can be suitablyselected from known administration methods basically according to eachanti-cancer agent. The anti-cancer agent can be administered accordingto the administration method, dose, and method of application describedin instructions for using each anti-cancer agent. When combined use ofthe anti-cancer agent with the sensitizer can promote reduction, in sizeof a tumor, the dosage and frequency of administration of theanti-cancer agent can be reduced.

II. Method For Treating Tumor

Another embodiment of the present invention provides a method oftreating a tumor comprising (1) locally administering a sensitizercomprising (a) hydrogen peroxide and (b) a hydrogel comprising acrosslinked gelatin gel to a tumor area, and (2) irradiating the tumorarea with radiation or administering an anti-cancer agent to a patientsuffering from the tumor.

The composition of the sensitizer used in the method of treating atumor, the method for applying the sensitizer, and methods of performingradiotherapy or anti-cancer chemotherapy are in accordance with thosedescribed in “I. Sensitizer for Enhancing Therapeutic Effect ofRadiation or Anti-cancer agent on Tumor.”

III. Composition For Treating Tumor Using Radiation or Anti-Cancer Agent

Another embodiment of the present invention is a composition fortreating a tumor by using radiation or an anti-cancer agent, thecomposition comprising a combination of (a) hydrogen peroxide and (b) ahydrogel comprising a crosslinked gelatin gel, and the composition beinglocally administered to a tumor area.

This composition is the same as the sensitizer described in “I.Sensitizer for Enhancing Therapeutic Effect of Radiation and Anti-canceragent on Tumor” and can be produced by using the same method, as themethod for producing the sensitizer. The method for applying thecomposition is also the same as for the sensitizer.

IV. Use in Manufacture of the Sensitizer For Increasing the Sensitivityto the Therapeutic Effect of Radiation and Anti-Cancer Agent on Tumor

Another embodiment of the present invention is use of a composition forlocal administration to a tumor area in the manufacture of a sensitizerfor enhancing a therapeutic effect of radiation or an anti-cancer agenton a tumor, the composition comprising a combination of the followingcomponents (a) and (b):

-   -   (a) hydrogen peroxide; and    -   (b) a hydrogel comprising a crosslinked gelatin gel.

The above (a) hydrogen peroxide and (b) hydrogel comprising acrosslinked gelatin gel according to this embodiment may be the same asthose described in “I. Sensitizer for Enhancing Therapeutic Effect ofRadiation or Anti-cancer agent on Tumor.” The method for producing thesensitizer and the method for applying the sensitizer may also be inaccordance with those described in “I. Sensitizer for EnhancingTherapeutic Effect of Radiation or Anti-cancer agent en Tumor.”

EXAMPLES

The present invention, is described below in more detail with referenceto Examples and Formulation Examples. However, the present invention isnor limited by the Examples, etc. In this specification, % means w/v %unless otherwise specified.

The animal experiments described in the Examples below were performedwith the approval of Koehi University Institutional Animal Care and UseCommittee in the Institute tor Laboratory Animal Research in theDepartment of Biofunctional Materials, Science Research Center, of KoehiUniversity.

Formulation Example 1 of Sensitizer

10 μl of an aqueous hydrogen, peroxide solution (about 3 w/v %) wasdirectly added dropwise per mg of MedGel (registered trademark)particles (PI5-95MS: a crosslinked gelatin hydrogel prepared fromgelatin with an isoelectric point of 4.8 to 5.0), and was tappedlightly. The mixture was allowed to stand at 37° C. for 1 hour to fullyimpregnate MedGel with the aqueous hydrogen peroxide solution, and theMedGel impregnated with the aqueous

hydrogen peroxide solution was floated on 50 μl of PBS. In this case,the final concentration of hydrogen peroxide was about 0.5 w/v %, andthe final concentration of MedGel was 1.6 w/v %.

The resulting sensitizer contained MedGel (crosslinked gelatin hydrogel)in an amount of 2 mg, on a dry weight basis, per 100 μl of thesensitizer,

Formulation Example 2 Sensitizer

10 μl of an aqueous hydrogen peroxide solution (about 0.5 w/v %) wasdirectly added dropwise per mg of MedGel (registered trademark)(PM-SF01: a crosslinked gelatin hydrogel prepared from gelatin with anisoelectric point of 8.0 to 9.0). The mixture was then allowed to standat 37° C. for 1 hour to fully impregnate MedGel with the aqueoushydrogen peroxide solution (about 0.5 w/v %). The aqueous hydrogenperoxide (about 0.5 w/v %) was prepared by diluting an aqueous hydrogenperoxide solution (about 3 w/v %) 6-fold with PBS, and used. In thiscase, the final concentration of hydrogen peroxide was about 0.5 w/v %,and the final concentration of MedGel was 1.6 w/v %.

Comparative Example 1

Hydrogen peroxide was diluted with PBS to a final concentration of 0.5w/v %.

Comparative Example 2

Immediately before use, 0.5 ml of an about 3 w/v % aqueous hydrogenperoxide solution was added to a syringe containing 25 mg of ahyaluronic acid formulation containing sodium hyaluronate in aconcentration of 1 w/v % (trade name ARTZ Dispo (registered trademark),produced by Seikagaku Corporation, containing 25 mg of sodiumhyaluronate per syringe (2.5 ml), and further containing 2.5 mg ofL-methionine, sodium chloride, disodium hydrogen phosphate, crystallinesodium dihydrogenphosphate, and an isotonizing agent; colorlesstransparent viscous aqueous liquid, pH: 6.8 to 7.8, osmotic pressureratio: 1.0 to 1.2 (ratio to the physiological saline), weight averagemolecular weight: 600,000 to 1,200,000) and the resulting mixture wasmixed well to prepare a radiation sensitizer or anti-cancer chemotherapysensitizer according to the present invention. The concentration ofsodium hyaluronate and the concentration of hydrogen peroxide in thissensitizer were 0.83 w/v % and about 0.5 w/v %, respectively.

Example 1 Comparison Between the Hydrogel Method and the KORTUC Methodin Effect 1. Method

Using 7-week old C3H/He female mice as experimental animals, anexperiment was performed in an animal experimentation facility. The micewere kept in a facility that was maintained at room temperature and at aconstant humidity and in which food and water were freely available.

SCCVII tumor cells (1.0×10⁵ cells/animal) were implanted subcutaneouslyinto the right lower thigh of the mice. The mice were grouped asfollows:

Group 1: Control group (receiving the sensitizer of Comparative Example1)

Group 2: KORTUC group (receiving the sensitizer of Comparative Example2)

Group 3: MedGel group (receiving the sensitizer of Formulation Example1)

At the stage of a tumor diameter of about 1 cm, the sensitizersobtained, in Comparative Example 1, Comparative Example 2, andFormulation Example 1 were injected into the tumor areas. For theinjection, an insulin syringe (with a 26-gauge needle) was used, andeach sensitizer was injected in an amount of 200 μl . 72 hours after theadministration of the sensitizer, electron beams with an energy of 6 MeVwere irradiated with 30 Gy using a linear accelerator (EXL-20TP,produced by Mitsubishi Electric, Tokyo). When irradiated, the mice wereanesthetized and irradiated using a mouse-immobilizing device. (FIG. 1).This device is for locally irradiating a leg portion in mice andprotecting the whole body other than the leg portion having a tumor byusing a copper plate with a thickness of 4.5 mm.

With the observation period set to 60 days, changes in tumor volume andsurvival rate were then checked. The tumor volume was observed using aVernier caliper.

The tumor volume (V) was obtained by measuring the minor axis (W) andmajor axis (L) of the tumor with a Vernier caliper and calculating thetumor volume by the following formula: v=(W2×L) /2.

2. Results

As shown in FIG. 2, mice in the MedGel group had the highest survivalrate, and their survival rate after 60 days was 80%. The control grouphad a survival rate of 60%. The KORTUC group had a survival rate of 50%.If one animal that died from anesthesia when irradiated is excluded, thesurvival rate of the KORTUC group is 60%, which is the same as thesurvival rate of the control group.

The irradiation in this experiment was performed 72 hours after locallyinjecting each drug into the tumor area. The conventional KORTUC canmaintain a partial pressure of oxygen in tumor tissue for 24 hours.Therefore, it is thought that in this experiment, the partial pressureof oxygen in tumor tissue had already decreased in the KORTUC groupbefore the irradiation with radiation, and sufficient therapeuticeffects were therefore not obtained.

The results suggest that the hydrogel comprising a crosslinked gelatingel according to the present invention can maintain the partial pressureof oxygen in tumor tissue not only immediately after the administrationof sensitizers but also 72 hours after the administration.

With respect to changes in tumor volume, the tumor growth rate wasmostly greatly inhibited in the MedGel group (FIG. 3). In themeasurement on day 30 from the start of the measurement, the averagetumor volume in the MedGel group was equal to half or less than, halfthe volume in the other groups (FIG. 4). The MedGel group had two micein which tumors were determined to have disappeared. One of the mice wasdetermined to he tumor-free on day 9 from the start of the measurement.Another was determined to be tumor-free on day 20. However, the mousewhich was determined to be tumor-free on day 20 actually had contractureat the joint and no tendency of tumor growth was observed; therefore,the mouse was determined to have not a tumor but a contracture at thejoint. However, since tendency for tumor growth was observed, later, themeasurement was started at that point in time. The control group alsohad a mouse in which a tumor was determined to have disappeared.However, since tendency for tumor growth was observed again, it wasdetermined to be a tumor and the measurement was started at that pointin time.

The results of this experiment suggest that the hydrogel comprising thecrosslinked gelatin gel of the present invention is the most effectivesubstrate in terms of maintaining oxygen partial, pressure in tumortissue, compared to the KORTUC group. The MedGel group had the highestsurvival rate, and disappearance of tumors was visually observed in someof the mice, and a therapeutic effect of almost complete response wasobserved. These results were obtained presumably because using acombination of hydrogen peroxide and a hydrogel comprising a crosslinkedgelatin gel as a sensitizer can increase the sensitivity to radiotherapyand thus enhance tumor inhibitory effects.

Example 2 Time-Dependent Change in the Form of MedGel

MedGel was mixed with an aqueous hydrogen peroxide solution (oxydol)according to Formulation Example 1, and time-dependent changes in theform of MedGel were observed (FIG. 5).

Even after 10 days, MedGel particles were not dissolved and MedGelmaintained its form.

This result suggests that release of hydrogen peroxide by MedGelcontinues during radiotherapy,

Example 3 Effect on Tumor Volume of Irradiation With Radiation Performed48 Hours After Administration of the Sensitizer to Tumor 1. Method

SCCVII tumor cells (1.0×10⁵ cells/animal) were implanted subcutaneouslyinto the lower-right thigh of the mice in the same manner as in the “1.Method” section of Example 1. The mice were grouped as follows:

Group 1: Control group (receiving no sensitizer)

Group 2: KORTUC group (receiving 100 μl of the sensitizer of ComparativeExample 2)

Group 3: P15-100 group (receiving the sensitizer of Formulation Example1 in an amount of 2 mg, on a dry weight basis of MedGel) Group 4: PI5-30group (receiving the sensitizer of Formulation Example 1 in an amount of0.6 mg, on a dry weight basis of MedGel)

Group 5: PI9-2 group (receiving the sensitizer of Formulation Example 2in an amount of 2 mg, on a dry weight basis of MedGel)

Group 6: PI9-0.6 group (receiving the sensitizer of Formulation Example2 in an amount of 0.6 mg, on a dry weight basis of MedGel)

At the stage of a tumor volume of about 200 to 250 mg, 100 μl of thesensitizer of Comparative Example 2, 100 μl of the sensitizer ofFormulation Example 1 (in an amount of 2 mg, on a dry weight basis ofMedGel), or 30 μl of the sensitizer of Formulation Example 1 (in anamount of 0.6 mg, on a dry weight basis of MedGel) was injected intotumors. Because the sensitizer of Formulation Example 2 was in the formof an elastic solid, the tumor area was incised and the sensitizer wasimplanted in an amount of 2 or 0.6 mg, on a dry weight basis of MedGel(crosslinked gelatin gel), per about 200 to 250 mg of the tumor volume.

Forty-eight hours after the administration of the sensitizer, tumorcells were irradiated with radiation under the same condition as in the“1. Method” section of Example 1, and the tumor volume on day 37 fromthe administration of the sensitizer (on day 35 from the irradiation)was measured.

2. Results

As shown in FIG. 6, no substantial difference was observed in tumorvolume among the KORTUC group (KORTUC), PI9-2 group (PI9-2), and thecontrol group (control). In contrast, a tumor volume increase wasinhibited in the PI5-100 group (PI5-100), PI5-30 group (PI5-30), andPI9-0.6 group (PI9-0.6), compared to the KORTUC group. In particular, inthe PI5-30 group and PI9-0.6 group, this inhibition effect was great.These results suggest that the hydrogel comprising a crosslinked gelatingel is preferably locally administered to the tumor area in an amount ofabout 0.1 to 1 mg, on a dry weight basis of the crosslinked gelatin gel,per 200 mm³ of the tumor volume. This data amount corresponds to a humandose of 0.1 to 5 mg or 0.5 to 10 mg.

Example 4 Effect on Tumor Volume of Irradiation With Radiation Performed72 Hours After Administration of the Sensitizer to Tumor 1. Method

SCCVII tumor cells (1.0×1.0⁵ cells/animal) were implanted subcutaneouslyinto the right lower thigh of the mice in the same manner as in the “1.Method” section, of Example 1. The mice were grouped as follows:

Group 1: Control group (receiving no sensitizer)

Group 2: KORTUC group (receiving 100 μl of the sensitizer of ComparativeExample 2)

Group 3: PI5-30 group (receiving the sensitizer of Formulation Example 1in an amount of 0.6 mg, on a dry weight basis of MedGel)

Group 4: PI9-0.6 group (receiving the sensitizer of Formulation Example2 in an amount of 0.6 mg, on a dry weight basis of MedGel)

At the stage of a tumor volume of about 200 to 250 mg, 100 μl of thesensitizer obtained in Comparative Example 2, or 30 μl of the sensitizerobtained in Formulation Example 1 was injected into each tumor. Becausethe sensitizer obtained in Formulation Example 2 was in the form of anelastic solid, the tumor area was incised and the sensitizer wasimplanted in an amount of 2 or 0.6 mg, on a dry weight basis of MedGel(crosslinked gelatin gel), per about 200 to 250 mg of the tumor volume.

Seventy-two hours after the administration of the sensitizer, the tumorwas irradiated with radiation under the same conditions as in the “1.Method” section of Example 1, and the tumor volume on day 37 from theadministration of the sensitizer (on day 34 front the irradiation withradiation) was measured.

2. Results.

As shown in FIG. 7, no substantial difference was observed in tumorvolume among the KORTUC group (KORTUC), PI9-0.6 group (PI9-0.6), and thecontrol group (control). In contrast, an increase in tumor volume wasinhibited in the PI5-30 group (PI5-30), compared to the other groups.These results suggest that a hydrogel comprising a crosslinked gelatingel that is prepared from a gelatin having an isoelectric point of 4.5to 5.5 is preferably used.

1-11. (canceled)
 12. A sensitizer for enhancing a therapeutic effect ofradiation or an anti-cancer agent on a tumor, the sensitizer comprisinga combination of: (a) hydrogen peroxide and (b) a hydrogel comprising acrosslinked gelatin gel prepared from an acidic gelatin, and thesensitizer being locally administered to a tumor area.
 13. Thesensitizer according to claim 12, wherein the acidic gelatin has anisoelectric point of 4.5 to 5.5.
 14. The sensitizer according to claim12, wherein the tumor is resistant to treatment using radiation or ananti-cancer agent.
 15. The sensitizer according to claim 12, wherein afirst administration of the sensitizer is performed during a period from3 days before a first irradiation with radiation or a firstadministration of the anti-cancer agent until the start of theirradiation or the administration of the anti-cancer agent.
 16. Thesensitizer according to claim 15, wherein a second administration of thesensitizer is performed 5 to 14 days after the first administration ofthe sensitizer.
 17. The sensitizer according to claim 12, wherein thecrosslinked gelatin gel has a water content of 92 to 99.7%.
 18. Thesensitizer according to claim 12, which, in the form of a finalformulation, is administered in an amount of 0.1 to 10 mg, on a dryweight basis of the crosslinked gelatin gel, per 200 mm³ of the tumorvolume.
 19. The sensitizer according to claim 12, which, in the form ofthe final formulation, contains hydrogen peroxide in an amount of 0.01to 3.5 wt. %.
 20. A method for treating a tumor, comprising the stepsof: locally administering to a tumor area a sensitizer comprising acombination of (a) hydrogen peroxide and (b) a hydrogel comprising acrosslinked gelatin gel prepared from an acidic gelatin; and (2)irradiating the tumor area with radiation or administering ananti-cancer agent to a patient suffering from the tumor.
 21. Acomposition for treating a tumor using radiation or an anti-canceragent, the composition comprising a combination of (a) hydrogen peroxideand (b) a hydrogel comprising a crosslinked gelatin gel prepared from anacidic gelatin, and the composition being locally administered to atumor area.
 22. Use of a composition for local administration to a tumorarea in the manufacture of a sensitizer for enhancing a therapeuticeffect of radiation or an anti-cancer agent on a tumor, the compositioncomprising a combination of the following components (a) and (b): (a)hydrogen peroxide; and (b) a hydrogel comprising a crosslinked gelatingel prepared from an acidic gelatin.